Human cytomegalovirus (HCMV) is the prototypical member of the beta-herpesvirus family. Epidemiological studies have shown that HCMV infection is widespread. In healthy individuals infection is generally asymptomatic, but the virus can cause serious disease in people with immature or compromised immune systems. It is the leading infectious disease cause of birth defects (primarily hearing loss) and a life- threatening adventitious agent in transplant recipients. The long-term objective of this research program is to elucidate the function of HCMV genes that regulate the interaction of the virus with its host cell and thereby control viral replication and pathogenesis. This proposal will study HCMV immediate-early gene products. These proteins and RNAs have the potential to exert profound effects on the virus-host interaction, because they are present at the very start of the infectious process. This is a renewal application that builds on our recent progress, which indicates that the UL123-coded IE1 protein controls the acetylation of proteins, reveals that the IRS1/TRS1 proteins have profound effects on the expression of viral mRNAs, and demonstrates that the so-called 5kb RNA is a stable intron. I now propose to extend these observations. My specific aims investigate the function and biochemical activities of (1) the HCMV UL122-coded IE1 protein; (2) the HCMV IRS1/TRS1 proteins; and (3) the HCMV 5kb transcript domain and its MCMV orthologue, the 7.2kb transcript domain. This project will include analysis of a laboratory strain (AD169) and a clinical isolate (VR1814, FIX) of HCMV. HCMV replication will be studied in fibroblasts, the cell commonly used to propagate HCMV; endothelial cells and epithelial cells.